SPERM GLYCINE RECEPTORS AND ZONA-INITIATED EXOCYTOSIS

描述(由申请人提供)：哺乳动物的顶体反应(AR)是精子头部的一种胞吐事件，对受精至关重要。哺乳动物精子中存在神经元甘氨酸受体/氯通道(GlyR)，是卵子透明带(ZP)蛋白ZP3启动AR所必需的。这项拨款的总体目标是增加我们对GlyR在ZP启动的AR中的作用以及控制其激活的机制的了解。我们假设ZP直接或通过受体串扰激活GlyR，通过GlyR的氯离子外流至少是导致AR所必需的钙内流的去极化的部分原因，GlyR的激活涉及PKA介导的磷酸化。分别用成像法、光度法和拮抗剂体外AR法研究小鼠ZP和人重组人ZP3(RhZP3)对小鼠精子和人精子的作用。具体目标如下。1.人和小鼠精子GlyR是否介导了重组人ZP3、小鼠ZP或甘氨酸在AR过程中引起的细胞内Ca~(2+)升高。2.人和小鼠精子GlyR是否参与甘氨酸、重组人ZP3或小鼠ZP引起的膜电位变化。3.确定蛋白激酶A(PKA)对GlyR的磷酸化是否参与了甘氨酸、小鼠ZP或rhZP3引发的AR。4.确定PKA是否在GlyR介导的膜电位变化以及甘氨酸、小鼠ZP和rhZP3引起的钙离子浓度升高中起重要作用。5.检测GlyR突变小鼠精子中ZP介导的膜电位和Ca~(2+)i变化是否消失或降低。6.体外检测GlyR突变小鼠精子的AR是否发生在完整小鼠卵子的ZP上。如果不是，确定这些精子在ZP上时，AR和受精是否受到孕酮的刺激？将人精子和重组人ZP3与小鼠精子和小鼠体内合成ZP的结果进行比较，将有助于我们确定哺乳动物是否存在共同的机制。

DESCRIPTION (provided by applicant): The mammalian acrosome reaction (AR) is a sperm head exocytotic event essential to fertilization. A neuronal glycine receptor/Cl channel (GlyR) is present in mammalian sperm and is necessary for the AR initiated by the egg zona pellucida (ZP) protein ZP3. The overall goal of this grant is to increase our understanding of the role of the GlyR in the ZP-initiated AR and the mechanisms involved in controlling its activation. We hypothesize that ZP activates the GlyR directly or via receptor cross-talk, that Cl- efflux through the GlyR is at least partly responsible for depolarization leading to Ca 2+ influx essential to the AR and that activation of the GlyR involves PKA-mediated phosphorylation. The effects of mouse ZP and human recombinant human ZP3 (rhZP3) on mouse sperm and human sperm respectively will be studied using imaging, photometry and antagonists in in vitro AR assays. The following are the Specific Aims. 1. Determination of whether the human and mouse sperm GlyR mediate the intracellular Ca 2+ (Ca2+i) increase elicited by rhZP3, mouse ZP or glycine during the AR. 2. Determination of whether the human and mouse sperm GlyR are involved in the membrane potential change elicited by glycine, rhZP3 or mouse ZP. 3. Determination of whether phosphorylation of the GlyR by protein kinase A (PKA) is involved in AR initiated by glycine, mouse ZP or rhZP3. 4. Determination of whether PKA is important to GlyR mediation of membrane potential change and to the Ca2+i increase elicited by glycine, mouse ZP and rhZP3. 5. Determination of whether mouse ZP-mediated membrane potential and Ca2+i changes would be absent or decreased in sperm from mice with GlyR mutations. 6. Determination of whether the AR of sperm from mice with GlyR mutations occur on the ZP of intact mouse eggs in vitro. If not, determination of whether the AR and fertilization be stimulated by progesterone in these sperm while they are on the ZP? Being able to compare results obtained with human sperm and rhZP3 and those obtained with mouse sperm and mouse ZP synthesized in vivo will help us determine whether common mechanisms are involved in mammalian species.